Direct continuous fluorometric assay for monoamine oxidase B.

The first direct and continuous fluorometric assay for monoamine oxidase B (MAO B) has been developed. E-2,5-Dimethoxycinnamylamine hydrochloride was designed and synthesized and was found to be an excellent substrate for MAO B (Km = 218 microM, Kcat = 435 min-1). This compound has an intense purple fluorescence when irradiated at lambda ex = 343 nm (lambda em = 393 nm) in Tris buffer, pH 9.0, or sodium phosphate buffer, pH 7.2, but under the same conditions, the corresponding aldehyde, the product of the MAO-catalyzed oxidation of E-,5-dimethoxycinnamylamine hydrochloride, does not fluoresce. The activity of MAO B, therefore, can be determined efficiently and rapidly by continuously following the decrease in fluorescence at 393 nm at enzyme concentrations as low as 100 nM. The change in fluorescence is linear up to a substrate concentration of 500 microM.